The Central Dogma of Molecular Genetics is “DNA makes RNA makes protein” (Lehninger, 1975). Information in biological systems flows from DNA to RNA to protein. This is shown in the following scheme;

In addition, double-stranded DNA can be used as a template to make new DNA. In nature, DNA is usually synthesized from DNA in a process called DNA replication.

In nature, DNA can also be formed from an RNA template. This process, called reverse transcription, is catalyzed by viral enzymes known as reverse transcriptases.

Notably, there is no known natural synthesis of RNA (or DNA) from a protein template, which could be considered “reverse translation”. To be more precise, in this document the term poly-amino acid reverse translation (PAA-RT) is used to describe the process shown (see definitions, below).

Not only has PAA-RT not been found in nature, it has not been carried out in the laboratory.
All of the processes described above, with the exception of PAA-RT, occur in nature. In addition, all of the processes described above, again with the exception of PAA-RT, are used in the biological research and development laboratory both in vivo and in vitro. In vivo applications involve the use of cells, commonly the bacterium E. coli, to produce protein from DNA, for example. In vitro studies often involve isolated enzymes. For example, transcription can be carried out using commercial kits (e.g., Promega Corp., Madison, Wis.). Similarly, reverse transcriptases, usually from avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MMLV), are used for reverse transcription (e.g., CLONTECH, Palo Alto, Calif.; Life Technologies, Inc., Rockville, Md.; New England Biolabs, Beverly, Mass.). In vitro translation can be carried out by eukaryotic (using rabbit reticulocyte lysates or wheat germ lysates) or prokaryotic (E. coli) systems (e.g., Promega Corp., Madison, Wis.). In addition, translation can be carried out using a novel system in which the encoding RNA and the growing peptide are linked in one chain (Phylos, Inc., Lexington, Mass.). Despite its high potential value, no reports of in vivo or in vitro PAA-RT exist.
Historically, there have been published speculations that PAA-RT may have existed in nature in molecular evolution, and indeed may exist undiscovered in nature today (Mekler, 1967; Cook, 1977; Craig, 1981; Biro, 1983; Trevors, 2001). All of these papers speculate on whether or not natural PAA-RT could have taken place and do not propose that PAA-RT could be carried out in the laboratory.
Two U.S. patents refer to reverse translation. One, Apparatus For Reverse Translation (U.S. Pat. No. 4,551,797), refers to data storage and has nothing to do with proteins or biotechnology. The other, Systematic Polypeptide Evolution By Reverse Translation (U.S. Pat. No. 5,843,701), uses the term reverse translation, but that invention does not describe a means of directly translating a peptide/protein into an oligo/polynucleotide. Instead, it describes a means of amplifying a peptide by using a covalent conjugate of the said peptide and its (already-synthesized) encoding mRNA. In the invention, the peptide portion of the peptide-RNA conjugate is captured and the mRNA is translating to form multiple copies of the peptide.
Recently a paper was published in which it was speculated that PAA-RT could be carried out in the laboratory (Nashimoto, 2001). The author suggested that PAA-RT might be carried out using designed RNA molecules and enzymes. An RNA molecule was designed and produced that bound the free amino acid, arginine, and the arginine-encoding codon, AGG. A self-cleavage reaction transferred the AGG codon to an acceptor RNA molecule. A number of critical steps in RT were not described. For example, not addressed or enabled were; how binding of the amino acid triggers RNA synthesis, how the peptide/protein would be “read” in sequence, and how the folded protein would be unfolded for PAA-RT to take place. In addition, PAA-RT “tools” (designed RNA molecules and specific enzymes) are required for each of the 20 amino acids, but the chemistry and/or molecular biology for making them is not disclosed in a way that would enable one skilled in the art to accomplish PAA-RT (only an arginine/AGG-binding RNA is disclosed). This is in stark contrast to the present invention, which describes PAA-RT in detail sufficient for one skilled in the art to perform the invention.
The present invention is the first description of a method of PAA-RT. The key component of the disclosed in vitro method for carrying out PAA-RT is the use of a so-called “Reverse Translation Element” (RTE). Amino acid derivatives are sequentially released from peptides/proteins. RTEs promote the exchange of these freed amino acid derivatives for their corresponding encoding trinucleotide codons. The codons are then sequentially coupled to the nascent peptide-encoding polynucleotide chain.
Until now, the synthesis of an encoding polynucleotide (RNA or DNA) that encodes a specific peptide or protein involved more than one process. Conventionally, a purified peptide or protein is sequenced using an automated amino acid sequencing machine. Following sequencing, the identity and order of the amino acids are read. From the sequence, an oligonucleotide is synthesized using a second instrument, an oligonucleotide synthesizer. Oligos may also be synthesized manually. From the prepared oligo, the full-length polynucleotide can be cloned. From the full-length polynucleotide, the protein can be produced. Drawbacks of this, the state of the art, procedure include; (1) it is time consuming, and (2) sufficient purified protein must be obtained for accurate amino acid sequencing.